The threat of engineered bacterial bioweapons that are resistant to current antibiotic treatments provides a powerful mandate to develop novel classes of therapeutic compounds. This proposal presents a concerted effort of three complementary groups at the Rockefeller University (Dr. Vincent Fischetti, Dr. Alexander Tomasz, and Dr. Erec Stebbins) to identify novel inhibitors of the Category A pathogens, B. anthracis and Y. pestis, as well as Category B food and water safety threats. The first and central research project is based on the observation that resistance to bacteriophage lytic enzymes, such as PlyG for B. anthracis, is an extremely rare event, from which we hypothesize that the binding domain of these enzymes targets a critical component in the bacterial cell wall to assure phage release (in a sense through evolution, the phage have performed the high throughput assay to identify this component). Thus, the pathway to the synthesis of this wall component represents a target for antibiotic development. The core project will therefore focus on the identification, isolation and characterization of the lytic enzyme binding substrate. Once identified, the pathway towards its synthesis will be determined. Those enzymes catalyzing the pathway will be isolated, purified and crystallized, and inhibitors identified. The cell wall structural experience of Alex Tomasz, feeds into the core project but also broadens it to a search for additional novel targets against B. anthracis by high resolution analysis of the cell walls and by the isolation of conditional lethal mutants - using in vivo transposition of a mariner type element. Cell walls occupy center stage in his project since (i) the target of PlyG is in the wall; (ii) cell walls play an important role in the devastating septic shock caused by B. anthracis infections and (iii) the genome of B. anthracis contains wall-related determinants that are virulence factors in other bacteria. Erec Stebbins will provide the expertise in protein structure and crystallization along with the identification of small molecule inhibitors for these structures. His project expands the novel therapeutics search to include Y. pestis and Category B pathogens through a translational component in computation drug discovery coupled to structural determination. In total, these efforts target central virulence factors in critical biowarfare pathogens.